RESUMEN
PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.
Asunto(s)
Empalme Alternativo , Enfermedad de Charcot-Marie-Tooth , Enfermedades Mitocondriales , Humanos , Empalme Alternativo/genética , Masculino , Femenino , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Niño , NADH Deshidrogenasa/genética , Linaje , Mutación/genética , Fenotipo , Secuenciación del Exoma , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Mitocondrias/genética , Mitocondrias/patología , Complejo I de Transporte de Electrón/genética , Adulto , Preescolar , AdolescenteRESUMEN
Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Tirosina-ARNt Ligasa , Animales , ADN Complementario/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Drosophila/genética , Mutación , Neuronas/metabolismo , Tirosina-ARNt Ligasa/metabolismo , Modelos Animales de Enfermedad , Mamíferos/genéticaRESUMEN
Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes. Charcot-Marie-Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies. A subtype of these diseases--CMT type 2D (CMT2D)--is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function. How mutations in GlyRS (GlyRS(CMT2D)) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRS(CMT2D) acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRS(CMT2D) to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRS(CMT2D) that antagonizes the VEGF-Nrp1 interaction, and indicate that the VEGF-Nrp1 signalling axis is an actionable target for treating CMT2D.
Asunto(s)
Unión Competitiva , Enfermedad de Charcot-Marie-Tooth/metabolismo , Glicina-ARNt Ligasa/metabolismo , Animales , Axones/enzimología , Axones/metabolismo , Axones/patología , Línea Celular , Supervivencia Celular , Enfermedad de Charcot-Marie-Tooth/tratamiento farmacológico , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Femenino , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/genética , Ligandos , Masculino , Ratones , Modelos Moleculares , Neuronas Motoras/enzimología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Destreza Motora/efectos de los fármacos , Mutación/genética , Neuropilina-1/deficiencia , Neuropilina-1/genética , Neuropilina-1/metabolismo , Unión Proteica , Multimerización de Proteína , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor A de Crecimiento Endotelial Vascular/uso terapéuticoRESUMEN
The increasing resistance of many pathogens to most of the common antimicrobials requires the development of new substances with more effective antimicrobial properties. In the present work, we investigated the mechanism of the antimicrobial activity of novel water soluble ammonium quaternary benzanthrone (Compound B) on model membranes, composed of dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine (DPPE). The lipids were chosen to represent a model of a bacterial membrane. The changes in surface pressure of the model membranes, before and after the addition of Compound B, were studied by the Langmuir's monolayer method, and the compressional modulus for each monolayer was determined. In addition, the surface morphology of the lipid monolayers before and after injection of Compound B was monitored by Brewster Angle Microscopy. The results showed that Compound B penetrated all the monolayers studied. The most noticeable effects were found with the negatively charged phosphatidylglycerols and with DPPE leading to the conclusion that the electrostatic interactions between the compound and the lipid head groups and the possible formation of hydrogen bonds between the amino group of the ethanolamine and the keto groups in the structure of Compound B are of great importance. In addition, the penetration ability of the benzoquinone with all phospholipids studied was stable even at higher values of the surface pressure, i.e. thicker monolayers, due to the hydrophobic interaction, which plays also an important role for the antimicrobial activity of Compound B.
Asunto(s)
Compuestos de Amonio , Antiinfecciosos/química , Antiinfecciosos/farmacología , Benzo(a)Antracenos/química , Benzo(a)Antracenos/farmacología , Compuestos de Amonio/química , Benzo(a)Antracenos/síntesis química , Membranas Artificiales , Estructura Molecular , Fosfatidilgliceroles/química , Fosfolípidos/química , Solubilidad , Propiedades de Superficie , Agua/químicaRESUMEN
Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Proteínas del Citoesqueleto/genética , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/genética , Adolescente , Alelos , Biopsia , Mapeo Cromosómico , Consanguinidad , Salud de la Familia , Femenino , Fibroblastos/metabolismo , Genes Recesivos , Ligamiento Genético , Marcadores Genéticos , Haplotipos , Humanos , Escala de Lod , Pérdida de Heterocigocidad , Malasia , Masculino , Mutación Missense , Neuronas/metabolismo , Linaje , Secuenciación del ExomaRESUMEN
BACKGROUND: Inherited peripheral neuropathies (IPNs) represent a broad group of genetically and clinically heterogeneous disorders, including axonal Charcot-Marie-Tooth type 2 (CMT2) and hereditary motor neuropathy (HMN). Approximately 60%-70% of cases with HMN/CMT2 still remain without a genetic diagnosis. Interestingly, mutations in HMN/CMT2 genes may also be responsible for motor neuron disorders or other neuromuscular diseases, suggesting a broad phenotypic spectrum of clinically and genetically related conditions. Thus, it is of paramount importance to identify novel causative variants in HMN/CMT2 patients to better predict clinical outcome and progression. METHODS: We designed a collaborative study for the identification of variants responsible for HMN/CMT2. We collected 15 HMN/CMT2 families with evidence for autosomal recessive inheritance, who had tested negative for mutations in 94 known IPN genes, who underwent whole-exome sequencing (WES) analyses. Candidate genes identified by WES were sequenced in an additional cohort of 167 familial or sporadic HMN/CMT2 patients using next-generation sequencing (NGS) panel analysis. RESULTS: Bioinformatic analyses led to the identification of novel or very rare variants in genes, which have not been previously associated with HMN/CMT2 (ARHGEF28, KBTBD13, AGRN and GNE); in genes previously associated with HMN/CMT2 but in combination with different clinical phenotypes (VRK1 and PNKP), and in the SIGMAR1 gene, which has been linked to HMN/CMT2 in only a few cases. These findings were further validated by Sanger sequencing, segregation analyses and functional studies. CONCLUSIONS: These results demonstrate the broad spectrum of clinical phenotypes that can be associated with a specific disease gene, as well as the complexity of the pathogenesis of neuromuscular disorders.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Atrofia Muscular Espinal/genética , Adulto , Anciano , Agrina/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Biología Computacional , Enzimas Reparadoras del ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/genética , Proteínas Musculares/genética , Atrofia Muscular Espinal/fisiopatología , Linaje , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores sigma/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Secuenciación del Exoma , Receptor Sigma-1RESUMEN
While having multiple aminoacyl-tRNA synthetases implicated in Charcot-Marie-Tooth (CMT) disease suggests a common mechanism, a defect in enzymatic activity is not shared among the CMT-causing mutants. Protein misfolding is a common hypothesis underlying the development of many neurological diseases. Its process usually involves an initial reduction in protein stability and then the subsequent oligomerization and aggregation. Here, we study the structural effect of three CMT-causing mutations in tyrosyl-tRNA synthetase (TyrRS or YARS). Through various approaches, we found that the mutations do not induce changes in protein secondary structures, or shared effects on oligomerization state and stability. However, all mutations provide access to a surface masked in the wild-type enzyme, and that access correlates with protein misinteraction. With recent data on another CMT-linked tRNA synthetase, we suggest that an inherent plasticity, engendering the formation of alternative stable conformations capable of aberrant interactions, links the tRNA synthetase family to CMT.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Tirosina-ARNt Ligasa/química , Tirosina-ARNt Ligasa/metabolismo , Sustitución de Aminoácidos , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Estabilidad de Enzimas/genética , Humanos , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Dispersión del Ángulo Pequeño , Proteína 28 que Contiene Motivos Tripartito , Tirosina-ARNt Ligasa/genética , Difracción de Rayos XRESUMEN
Recessive mutations in the gene encoding the histidine triad nucleotide binding protein 1 (HINT1) were recently shown to cause a motor-predominant Charcot-Marie-Tooth neuropathy. About 80% of the patients exhibit neuromyotonia, a striking clinical and electrophysiological hallmark that can help to distinguish this disease and to guide diagnostic screening. HINT1 neuropathy has worldwide distribution and is particularly prevalent in populations inhabiting central and south-eastern Europe. With 12 different mutations identified in more than 60 families, it ranks among the most common subtypes of axonal Charcot-Marie-Tooth neuropathy. This article provides an overview of the present knowledge on HINT1 neuropathy with the aim to increase awareness and spur interest among clinicians and researchers in the field. We propose diagnostic guidelines to recognize and differentiate this entity and suggest treatment strategies to manage common symptoms. As a recent player in the field of hereditary neuropathies, the role of HINT1 in peripheral nerves is unknown and the underlying disease mechanisms are unexplored. We provide a comprehensive overview of the structural and functional characteristics of the HINT1 protein that may guide further studies into the molecular aetiology and treatment strategies of this peculiar Charcot-Marie-Tooth subtype.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Neuropatía Hereditaria Motora y Sensorial/genética , Síndrome de Isaacs/genética , Miotonía/genética , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedad de Charcot-Marie-Tooth/epidemiología , Enfermedad de Charcot-Marie-Tooth/patología , Neuropatía Hereditaria Motora y Sensorial/epidemiología , Neuropatía Hereditaria Motora y Sensorial/patología , Humanos , Síndrome de Isaacs/epidemiología , Síndrome de Isaacs/patología , Miotonía/epidemiología , Miotonía/patología , Enfermedades del Sistema Nervioso Periférico/epidemiología , Enfermedades del Sistema Nervioso Periférico/patologíaRESUMEN
Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.
Asunto(s)
Acetiltransferasas/genética , Enfermedad de Charcot-Marie-Tooth/genética , Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Acetiltransferasas/metabolismo , Adolescente , Adulto , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/complicaciones , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Humanos , Discapacidad Intelectual/complicaciones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Mutación , Linaje , Adulto JovenRESUMEN
Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-µ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Exoma/genética , Modelos Moleculares , Mutación Missense/genética , Fenotipo , Adulto , Secuencia de Bases , Enfermedad de Charcot-Marie-Tooth/patología , Mapeo Cromosómico , Femenino , Haplotipos/genética , Humanos , Datos de Secuencia Molecular , Linaje , Mapeo de Interacción de Proteínas , Análisis de Secuencia de ADN , Nervio Sural/patologíaRESUMEN
OBJECTIVE: To identify the unknown genetic cause in a large pedigree previously classified with a distinct form of axonal Charcot-Marie-Tooth disease type 2G (CMT2G) and to explore its transcriptional consequences. METHODS: Clinical reevaluation of the pedigree was performed, followed by linkage analysis with the redefined disease statuses, and whole genome and exome sequencing. The impact of the mutation was investigated by immunoblotting and transcriptome sequencing. RESULTS: Thirteen affected individuals over 3 generations displayed mild and quiescent lower-limb axonal sensorimotor neuropathy. Magnetic resonance imaging (MRI) of lower-limb musculature systematically showed fatty atrophy in clinical and subclinical mutation carriers. We redefined the disease-linked region to chr9q31.3-q34.2 and subsequently identified a novel missense variant in the E3 ubiquitin-protein ligase LRSAM1 (p.Cys694Tyr). Unlike previous reports, we demonstrated in patients' lymphoblasts that the mutation does not influence overall protein levels of LRSAM1, nor of its ubiquitylation target TSG101. The mutation is associated with several transcriptional changes, including a significant upregulation of another E3 ubiquitin-protein ligase, NEDD4L, and of TNFRSF21, a key regulator of axonal degeneration. INTERPRETATION: Our findings demonstrate that the isolated genetic entity CMT2G is caused by a missense mutation in LRSAM1 and should be reclassified as CMT2P. MRI of lower-limb musculature can be used to detect minimal signs of the disease. Transcriptome analysis of patients' cells highlights novel molecular players associated with LRSAM1 dysfunction, and reveals pathways and therapeutic targets shared with amyotrophic lateral sclerosis and Alzheimer disease. Ann Neurol 2016;80:823-833.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación Missense , Ubiquitina-Proteína Ligasas Nedd4 , Conducción Nerviosa/genética , Conducción Nerviosa/fisiología , Linaje , Regulación hacia ArribaRESUMEN
We performed whole exome sequencing on a patient with Charcot-Marie-Tooth disease type 1 and identified a de novo mutation in PMP2, the gene that encodes the myelin P2 protein. This mutation (p.Ile52Thr) was passed from the proband to his one affected son, and segregates with clinical and electrophysiological evidence of demyelinating neuropathy. We then screened a cohort of 136 European probands with uncharacterized genetic cause of Charcot-Marie-Tooth disease and identified another family with Charcot-Marie-Tooth disease type 1 that has a mutation affecting an adjacent amino acid (p.Thr51Pro), which segregates with disease. Our genetic and clinical findings in these kindred demonstrate that dominant PMP2 mutations cause Charcot-Marie-Tooth disease type 1.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Proteína P2 de Mielina/genética , Adolescente , Exoma/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Conducción Nerviosa/genética , Linaje , Adulto JovenRESUMEN
PURPOSE: Homozygosity mapping is an effective approach for detecting molecular defects in consanguineous families by delineating stretches of genomic DNA that are identical by descent. Constant developments in next-generation sequencing created possibilities to combine whole-exome sequencing (WES) and homozygosity mapping in a single step. METHODS: Basic optimization of homozygosity mapping parameters was performed in a group of families with autosomal-recessive (AR) mutations for which both single-nucleotide polymorphism (SNP) array and WES data were available. We varied the criteria for SNP extraction and PLINK thresholds to estimate their effect on the accuracy of homozygosity mapping based on WES. RESULTS: Our protocol showed high specificity and sensitivity for homozygosity detection and facilitated the identification of novel mutations in GAN, GBA2, and ZFYVE26 in four families affected by hereditary spastic paraplegia or Charcot-Marie-Tooth disease. Filtering and mapping with optimized parameters was integrated into the HOMWES (homozygosity mapping based on WES analysis) tool in the GenomeComb package for genomic data analysis. CONCLUSION: We present recommendations for detection of homozygous regions based on WES data and a bioinformatics tool for their identification, which can be widely applied for studying AR disorders.Genet Med 18 6, 600-607.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad de Charcot-Marie-Tooth/genética , Proteínas del Citoesqueleto/genética , Paraplejía Espástica Hereditaria/genética , beta-Glucosidasa/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/patología , Mapeo Cromosómico , Consanguinidad , Femenino , Glucosilceramidasa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Masculino , Mutación , Linaje , Polimorfismo de Nucleótido Simple/genética , Paraplejía Espástica Hereditaria/diagnóstico , Paraplejía Espástica Hereditaria/patología , Secuenciación del ExomaRESUMEN
Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2SH3TC2, histidine-triad nucleotide binding protein 1HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Mutación , Mapeo Cromosómico , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Homocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas del Tejido Nervioso/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Proteínas/genéticaRESUMEN
Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.
Asunto(s)
Axones/enzimología , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Genes Dominantes/genética , Mutación/genética , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Axones/patología , Bioensayo , Células COS , Línea Celular Tumoral , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/metabolismo , Chlorocebus aethiops , Prueba de Complementación Genética , Heterocigoto , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Recombinantes , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Alineación de Secuencia , Tirosina-ARNt Ligasa/químicaRESUMEN
Charcot-Marie-Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Mutación/genética , Proteínas de Unión al GTP rab/genética , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Diferenciación Celular , Línea Celular Transformada , Dendritas/patología , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Laminopatías , Larva , Masculino , Actividad Motora/genética , Neuroblastoma/patología , Células Receptoras Sensoriales/patología , Transducción Genética , Proteínas de Unión a GTP rab7RESUMEN
Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for the viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth (CMT) disease. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YARS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/complicaciones , Enfermedad de Charcot-Marie-Tooth/genética , Glicina-ARNt Ligasa/genética , Mutación/genética , Enfermedades del Sistema Nervioso Periférico/etiología , Tirosina-ARNt Ligasa/genética , Animales , Animales Modificados Genéticamente , Enfermedad de Charcot-Marie-Tooth/patología , Dextranos , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Humanos , Masculino , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Fibras Nerviosas/fisiología , Neuronas/patología , Neuronas/fisiología , Enfermedades del Sistema Nervioso Periférico/genética , Retina/patología , Retina/ultraestructura , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/etiología , Degeneración Retiniana/genética , Rodaminas , Alas de Animales/patología , Alas de Animales/ultraestructuraRESUMEN
Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygous splice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases, we identified two additional families with rare deleterious biallelic variants in COX18 . All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>G variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. Our study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Our research has important implications for the diagnostic workup of CMT patients.
RESUMEN
Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.
Asunto(s)
Actinas , Tirosina-ARNt Ligasa , Animales , Humanos , Actinas/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Drosophila/genética , Glicina-ARNt Ligasa/genética , Mutación , ARN de Transferencia , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Línea Celular TumoralRESUMEN
Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous and four aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.